Chemical component analysis is extensively used in many and varied fields. However, the uses for such chemical component analysis have been limited due the requirement for special equipment, physical space for the equipment, and the amount of time necessary for preparing for and conducting the analysis. To address these problems, there is much interest in developing “micro-total analysis systems” (μ-TAS) having dimensions similar to a credit card. Such systems combine a separating device, mixing device, measuring device, and analyzing device on the same substrate. Ideally, in use, the μ-TAS can deliver the sample solution to the analysis equipment and then analyze the sample while requiring microliters of sample at most. Moreover, the μ-TAS can be operated using similarly small amounts of reagents. Such systems have uniform reaction temperatures, superior controllability, and are disposable improving safety and hygiene.
Analysis by μ-TAS is useful in a number of applications including, but not limited to, medical, industrial, agriculture, molecular, and forensic investigations. Examples of medical applications include but are not limited to, measurement and inspection of blood components and biochemical analysis, such as measurement and inspection of various types of proteins, hormones, and antibodies. Examples industrial applications include but are not limited to, component analysis in manufactured products, component analysis of waste-water and component analysis of raw materials. Examples of use in agriculture include but are not limited to, measurement of sugar content in vegetables and fruits and measurement of chemical/pesticide residues both in the environment and on produce. Examples for use with genetic analysis include but are not limited to decoding of genetic information for diagnosis and prevention of genetic diseases.
For chemical component analysis in solution, μ-TAS employs microfluidic devices in which an internal channel is immobilized with a cognitive agent, such as, for example, an enzyme for a reaction catalyst or antibodies, binding proteins and receptors for capturing desired substrates or analytes. Such cognitive agents can be used to react specifically with a desired analyte (detected substance). Currently, microfluidic devices using the above mentioned cognitive agents such as enzymes, antibodies, binding proteins, etc. are limited because only a single cognitive agent is immobilized in the channels of the microfluidic device. When used in chemical component analysis in solution and for which multiple cognitive agents, e.g. enzymes or antibodies, receptors, ligands or the like are used, the solution sample has to be multiply contacted and reacted with different and multiple immobilized cognitive agents. This requires several microfluidic devices immobilized with single cognitive agent, as mentioned above, that are linked by a connecting channel of each device to form a composite device; or by connecting the microfluidic devices and connecting the channels. The deficiencies of these approaches are the difficulty of use and maintenance, solution leak at the joints which requires substantially reinforced joint areas, increases space needs and larger amounts of sample and reagent. These deficiencies result in a large amount of time needed in constructing and connoting the various devices, large amount of time needed to perform the analysis as well as more costly devices cost of analysis time and less consistent results.
Several methods of fabricating micro channel devices. These include, methods of forming a resin layer on a substrate (JP nos. 2002-283293 and 2004-167607) by attaching a resin layer formed by laser on a silicon, glass, or ceramic substrate; methods of fabricating a channel using a photoresist and exposing it to ultraviolet light through a mask, then removing non-exposed parts (JP no. 2004-194652); methods of fabricating a channel by micro-discharge; and methods of mechanical fabrication (etching) using a hard material, e.g. diamond, as a tool for the micro-fabrication. Additionally, methods of micro-channel fabrication using a mould begins with coating the photoresist on silicon substrate, exposing it through a mask, and removing the photoresist to generate an embossed portion to form a mould for the micro-channel on silicone substrate, adding a mixture of polydimethyl siloxane (PDMS) and hardening material onto the mould, obtaining a groove of the channel as the mixture hardens, detaching the hardened layer off the mould, and attaching the hardened layer on a substrate, e.g. silicone or glass (JP no. 2004-296099).
As with methods of fabricating microfluidic devices, methods of immobilizing enzymes can also be performed in various ways. However, a method of immobilizing different cognitive agents, such as multiple enzymes or antibodies, onto a single channel in discrete positions for use in continuous or cascade reactions is not known. Currently, a screen printing technique can be used for immobilizing different enzymes onto the same substrate, but the accuracy of placement is limited to a range of 500 μm to 1 mm. This limitation is far in excess of what is required for μ-TAS analysis.
In instances of reactions such as catalytic reactions, binding reactions, or antigen-antibody reactions, that include multiple steps that need to be performed continuously or sequentially using multiple cognitive agents, e.g. enzymes, antibodies; the use of a microfluidic device in these instances should be capable of controlling the reaction, using only minimal amounts of reactants/reagents as well as providing high efficiency. Furthermore, in cases of cascade reactions occurring in a single micro-channel it is a necessity to provide controlled and reproducible environment. For example, in a small system, it is difficult to separate the 1st reaction zone, from the 2nd reaction zone. But, if one were capable of immobilizing different cognitive agents at different positions along the micro-channel, the product from the 1st reaction could be used in the 2nd reaction and so on. To achieve such cascade reactions, it is necessary to immobilize the cognitive agents in different positions along the channel path in a discrete and well-defined manner. At present, it is difficult to immobilize cognitive agents in different positions along a micro-channel path having a uniform channel width with accuracy better than 500 μm. However, the use of narrower channels would result less reagents used and also provide more consistent results, reproducible reaction in much less time.
Therefore, it is desirable to provide a microfluidic device having different cognitive agents immobilized in discrete positions along a micro-scale channel that would allow sequentially reaction to be performed thereon. In some instances, it would be further desirable to utilize a method of quantitative component analysis to analyze the results.